Summary Rhabdomyosarcoma (RMS) accounts for ~3% of all pediatric cancers. Unfortunately, the overall 5-year survival rate for children diagnosed with metastatic RMS is less than 30%. Sarcoma patients experience higher rates of morbidity and mortality than other cancer patients, and this is particularly evident in children. As a result of their therapies, 42% of childhood cancer survivors experience severe, disabling, or life threatening conditions (including secondary tumors). Thus, there is clearly a need to develop new, more targeted treatment strategies for pediatric tumors; treatments that inhibit tumor progression yet confer limited side effects. The pro-metastatic transcription factor SIX1 is overexpressed in RMS, where it is plays a critical role in metastasis. During embryonic development, Six1 promotes precursor cell activation and migration to enable proper formation of muscle, kidney and the inner ear. However, after development is complete, Six1 expression is silenced or reduced in most tissues. Thus, Six1 is upregulated in the setting of RMS, where it is known to be important for progression of the disease. These data suggest that gaining an understanding of the mechanisms controlling Six1 downregulation during muscle development may provide a novel means by which to target Six1 in the setting of RMS. Such targeting may be anticipated to inhibit the tumor, yet have limited toxicity due to the paucity of Six1 expression in adult tissue. Throughout embryogenesis and myogenesis, microRNAs (miRs) have been shown to coordinate complex temporal and tissue-specific patterns of protein expression, including regulation of many homeobox genes. In tumor models, miRs have been shown to inhibit RMS tumor growth, and miR-mediated downregulation of Six1 can prevent kidney tumor progression. In zebrafish, there are two orthologs of Six1, six1a and six1b. Our recently published data demonstrate that miR30a negatively regulates both six1a and six1b in zebrafish muscle development, and that miR30a-mediated downregulation of Six1 is required for proper muscle development to occur. Thus, in this proposal we will test the following Hypothesis: Upregulation of Six1 is required for RMS tumor onset and progression. We will further test whether zebrafish will be an ideal model to rapidly and inexpensively test whether inhibitors of Six1 (beginning with miR30a, but in the future adding in additional miRs or novel Six1 small molecule inhibitors) will inhibit RMS progression. Specific aims: 1) To investigate the role of the Six1 transcriptional complex in RMS initiation and progression in zebrafish models, and 2) To identify potential therapeutic options for RMS through downregulation of Six1. The second of these aims will test, as proof of principal, whether miR30a has the potential to inhibit RMS progression. This R21 mechanism, by allowing us to develop the zebrafish models, will set the stage for testing novel small molecule inhibitors that target the Six1 transcriptional complex in the context of a whole organism. As we are currently developing such inhibitors, these models will allow for more efficient screening of a large number of compounds in vivo than could be done in other model systems.